Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein in its entirety.
Exciting progress has been made in melanoma biology. In the last several years a model of human melanocyte transformation has emerged based on the combined results of human genetic studies, cell biology, molecular pathology and mouse modeling (Chin. Nat Rev Cancer 2003; 3:559-70). Initial studies that identified the autocrine production of basic fibroblast growth factor as an early event in melanocyte transformation have been augmented by recent studies identifying ERK activation in melanoma tissues (Cohen et al. Clin Cancer Res 2002; 8:3728-33) and cell lines (Satyamoorthy et al. Cancer Res 2003; 63:756-9), and most importantly by the discovery of frequent BRAF mutations in melanoma tissues (Gorden et al. Cancer Res 2003; 63:3955-7; Davies et al. Nature 2002; 417:949-54; Dong et al. Cancer Res 2003; 63:3883-5; Kumar et al. Clin Cancer Res 2003; 9:3362-8; Pollock et al. Nat Genet 2003; 33:19-20; Uribe et al. Am J Dermatopathol 2003; 25:365-70; Yazdi et al. Pigment Cell Res 2003; 16:580). Many melanomas possessing mutant BRAF alleles have also acquired extra copies of the these alleles (Maldonado et al. J Natl Cancer Inst 2003; 95:1878-90). BRAF acts downstream of RAS, and studies have demonstrated that simultaneous mutations in RAS and BRAF are extremely rare in melanoma (Gorden et al. 2003, supra; Davies et al. 2002, supra; Dong et al. 2003, supra; Kumar et al. 2003, supra; Pollock et al. 2003, supra), suggesting that BRAF mutations substitute for at least some of the oncogenic function of mutant RAS. Experimental studies have demonstrated that several BRAF mutations, especially the V600E hotspot mutation (which was originally designated V599E, but has been renumbered V600E in accordance with advances in the field; see Kumar et al. 2003 supra), which accounts for 90% of BRAF mutations in melanoma, activate the MAPK cascade and can transform fibroblasts in culture (Davies et al. 2002, supra; Dong et al. 2003, supra).
In the clinic the treatment of metastatic melanoma remains one of the most formidable challenges today. Survival is best predicted by tumor stage, with patients with distant metastases (Stage IV) having a median survival of only 6 to 9 months, and 5-year survival rates of only 1% to 5%. Five-year survival of patients with regional lymph node disease (Stage III) ranges from 13% to 69% depending upon the number of nodes involved, the volume of disease in the nodes (microscopic only vs. clinically palpable) and thickness and ulceration status of the primary tumor. In clinically localized, stage I/II disease, 5-year survival ranges from 45% to 95% depending upon tumor thickness and the presence of ulceration (Balch et al. J Clin Oncol 2001; 19:3622-34). Patients whose tumors are thicker than 1 mm are often referred for sentinel lymph node biopsy. This technique aims to detect microscopic lymph node involvement, thereby selecting patients who might benefit from a regional lymph node dissection. However, the benefit of regional lymph node dissection is still debated among oncologists, as patients eventually succumb to melanoma due to the hematogenous dissemination of tumor cells (Balch et al. Elective Lymph Node Dissection. In: Balch CM, Houghton A, Sober A, Soong R, eds. Cutaneous Melanoma. St. Louis: Quality Medical, 2003:379-395).
The development of tumor markers to better stratify patients for their risk of developing metastases is under active investigation. Although assessment of tumor markers and selection of treatment based on the results has been part of the standard of care in colon and breast cancer management for several years (Bast et al. J Clin Oncol 2001; 19:1865-78), no such markers exist for melanoma. Several papers have been published investigating growth control genes, extracellular matrix-degrading enzymes, adhesion/signaling molecules, angiogenic factors and immunoregulatory molecules (Hwu et al. Diagnosis of Stage IV Disease. In: Balch CM, Houghton A, Sober A, Soong S, eds. Cutaneous Melanoma. St. Louis: Quality Medical, 2003:523-546 and references therein). Many studies have shown promise, but none have moved past the preliminary stages of development into a clinically useful assay. Most of these studies are based on immunohistochemistry, and it is anticipated that inter-laboratory variability will be a major obstacle to the adoption of all but the most promising markers (McShane et al. Clin Cancer Res 2000; 6:1854-64). Moreover, techniques involving immunohistochemistry are not well suited to rapid screening approaches because they are time consuming, and require invasive procedures for sample isolation and a significant degree of technical expertise.
In view of the above, new methods for use in the accurate diagnosis, prognosis, and/or monitoring of patients with melanoma are urgently needed. The method of the present invention addresses this need.